PROJECT SUMMARY Project 2 In the mid-1990s, vaccine-related side effects prompted the replacement of the whole Pertussis (wP) vaccine by a new acellular Pertussis vaccine (aP). Unexpectedly, whooping cough cases have recently increased, particularly in teenagers. In the preliminary data, we compared individuals born before 1995 and vaccinated in infancy with wP, with individuals born in 1996 or later and vaccinated with aP in infancy. Activation Induced Marker (AIM) assays allowed examination of responses directly ex vivo, highlighting a Th2 vs Th1/Th17 polarization of pertussis (PT)-specific CD4+ T cells. Remarkably, we detected differences in response to a contemporary aP booster, even though the first aP or wP priming occurred more than 18 years previously. aP priming was associated with 1) increased IL-9 and TGF-? and decreased IL-17 production in response to PT epitopes ex vivo, and 2) defective ex vivo capacity to expand memory cells one to two months following a booster aP immunization and in vitro proliferation in response to PT epitopes. However, the biological significance of these observations is limited by the fact that only responses in blood are measured and whether the same phenotypic differences are observed in situ, in the lymphatic tissues where memory T cell reside and immune responses are orchestrated. Accordingly, we propose to define the molecular mechanisms underlying the differential IL-9/IL-17 polarization of donors originally primed with aP vs wP (Aim 1). We will define the cellular characteristics of IL-9+ PT-specific CD4+ T cells after aP vaccination and molecular mechanisms underlying their development. We will study the IL-17 signature seen in wP vaccine-primed individuals, by targeted flow cytometry and AIM studies, and perform single-cell RNA-seq with the PT-specific CD4+ T cells. In Aim 2, the mechanisms involved in differential proliferation will be addressed by blocking in vitro TGF-b related pathways, and downregulating the ANAPC2 and WDR1 genes, which were identified in the preliminary data. The cellular basis of the differential proliferation of donors originally primed with aP vs wP will be investigated by cell depletion experiments. Aim 3 will use Lymph Node (LN) Fine Needle Aspirates (FNA) to interrogate PT responses in individuals originally primed with either wP or aP to assess differential germinal center (GC) Tfh cell and GC B cell responses in LNs of vaccine recipients, define whether differential antibody isotype responses of wP- vs aP-primed individuals is due to biases in GC Tfh helper functions to B cells and determine if circulating CD4+ T cells correlate with the vaccine immune response in the draining LN, the primary immune response site. In conclusion, we believe that results from this study will illuminate molecular reasons for lesser aP efficacy and illustrate human CD4+ T cell biological principles generally applicable to vaccinology and immunology.